Fine particles containing rare earth element and fluorescent probe using the same

ABSTRACT

Fine particles whose excitation light is not UV light or the like which has negative effects on a subject to be analyzed. The excitation light is emitted stably, and has excellent light emitting efficiency. Also a fluorescent probe including: fine particles containing a rare earth element excited by light having a wavelength in a range of 500 nm to 2000 nm and thereby emit up-conversion emission; and a specific binding substance which binds to the fine particles containing a rare earth element.

TECHNICAL FIELD

The present invention relates to fine particles containing a rare earthelement which are excited by red light or infrared light and emit whatis called “up-conversion emission” and a manufacturing method of thefine particles. The present invention also relates to a fluorescentprobe labeled by the fine particles containing a rare earth element. Thefine particles containing a rare earth element and the fluorescent probeof the present invention can suitably be used in the fields of genediagnosis, immunodiagnosis, medicinal development, environmentaltesting, biotechnology, a fluorescent inspection, and the like.

BACKGROUND ART

Conventionally, in the fields of medicine and biology, a method in whicha fluorescent material composed of organic molecules is used as a markerand fluorescence generated by UV irradiation is measured by an opticalmicroscope or a photo detector. Well-known examples of such a methodinclude an antigen-antibody fluorescent method. In this method, anantibody, to which an organic fluorescent body capable of emittingfluorescence is bound, is used. As the antigen-antibody reaction isextremely highly selective, as is often compared to a relationshipbetween a key hole and a key. Therefore, it is possible to identify thelocation of the antigen based on the distribution of fluorescenceintensity.

As another example, there is a fluorescence-utilizing method using socalled DNA chips. When this testing method is employed for a purpose ofdetermining the base sequence of an unknown DNA, the scheme thereof isas follows. That is, by reacting what is called DNA chips in which alarge number of DNA (DNA fragments) having known base sequences arearranged in spots-like on a substrate, and DNA having an unknown basesequence which is an organic fluorescent body labeled subject to betested, the base sequence of the subject is determined by analyzing theposition, strength and the like of the fluorescent spots on the DNAchips.

However, the aforementioned conventional organic fluorescent body usefulfor fluorescent labeling has problems, that is, a problem such that theorganic fluorescent body is not stable in storage and at the time ofmeasuring fluorescence, and there is a possibility of deterioration.

In order to solve the problems as describe above, there has beenproposed a method of using CdSe nanoparticles (“SemiconductorNanocrystals as Fluorescent Biological Labels” Marcel Bruchez Jr. etal., p2013-2016, SCIENCE Vol. 281, 25 Sep. 1998; “Quantum DotBioconjugates for Ultrasensitive Nonisotopic Detection” Warren C. W.Chan and Shuming Nie. P2016-2018, SCIENCE Vol. 281, 25 Sep. 1998).However, in the above-mentioned method, as the excitation light is bluelight or UV light, there arises a problem that, when the subject to beanalyzed or detected is a living cell or a living tissue, the excitationlight damages the analyzing or detecting subject. Further, when thesubject to be analyzed or detected is DNA or a protein, there is apossibility that UV light damages molecules. Therefore, in this method,there is a possibility that determination of base sequences and activitysites with high precision is disturbed.

As particles which emit light by excitation light of a longerwavelengths, Si nanoparticles which emits two-photon excitation havebeen proposed (“Second harmonic generation in microcrystallite films ofultra small Si nanoparticles” APPLIED PHYSICS LETTERS VOLUME 77, NUMBER25 18 Dec. 2000). However, as this method is based on a mechanism oflight emitting by two-photon absorption, there arises problems in thatthe light emitting efficiency is poor and the detection precisiondeteriorates and that super fine particles of no larger than 1 nm arenecessary and thus the manufacturing process thereof is complicated.

DISCLOSURE OF THE INVENTION

The present invention has been achieved in consideration of the problemsdescribed above. A main object of the present invention is to provide:fine particles whose excitation light is not the one such as UV light orthe like which has negative effects on a subject to be analyzed, emitlight stably, and has excellent light emitting efficiency; a method formanufacturing the fine particles; and a fluorescent probe labeled withthe fine particles.

In order to achieve the aforementioned object, the present inventionprovides a fine particle containing a rare earth element, characterizedin that it is excited by light having a wavelength in a range of 500 nmto 2000 nm and thereby emits up-conversion emission. As the fineparticles containing a rare earth element of the present invention arefine particles containing a rare earth element which thus emitsup-conversion emission, there is no need to use UV light or blue lightas excitation light when the particles are employed as a fluorescentprobe. Therefore, there is no possibility of damaging biopolymer to beanalyzed. Further, there is no problem of lacking in stability instorage like organic fluorescent body. And further, the fine particleshave an advantage of high light emitting efficiency.

In the above mentioned invention, it is preferable that comprising: acore portion containing the rare earth element; and a functional shellportion modifying a surface of the core portion; wherein the functionalshell portion comprising at least a specific binding substance bindingsite which can be bound to a specific binding substance. By comprisingsuch functional shell portion, binding to a specific binding substanceis easily possible.

Further, in the above mentioned invention, it is preferable that thespecific binding substance binding site is at least one of, a site withcondensation reactivity, a site with addition reactivity, a site withsubstitution reactivity, or a site capable of effecting a specificinteraction. Such sites as described above can bind to the specificbinding substances more securely.

In the present invention, it is preferable that the fine particlescontaining rare earth element comprises an agglutinate preventing sitewhich prevents agglutination of the fine particles containing a rareearth element. For example, in the case of being in a liquid having ahigh salt concentration such as a body fluid, it can be used withoutagglutination.

In the above mentioned invention, it is preferable that the agglutinatepreventing site is at least one of site of, a site having hydrogenbonding, or a site with hydrating ability. By comprising an agglutinatepreventing site as described, agglutination of the fine particlescontaining a rare earth element can reliably be prevented.

Further, it is preferable that the functional shell portion comprises acore portion binding site, which binds to the core portion. Bycomprising such a core portion binding site, the binding of the coreportion and the functional shell portion is made stronger, andinconveniences such as peeling of the functional shell portion from thecore portion can be prevented.

Further, it is preferable that the core portion binding site is at leastone of, a site with a function to bind to a metal, a site withcondensation reactivity, a site with addition reactivity, or a site withsubstitution reactivity. By comprising the site as described, binding ofthe core portion and the functional shell portion can be conducted moresecurely.

In the above mentioned invention, it is preferable that the averageparticle diameter of the core portion is in a range of 1 to 100 nm. Itis because when polynucleotide, an antigen, an antibody or the like isused as a specific binding substance, the average particle diameter inthe aforementioned range is preferable.

Moreover, it is preferable that the core portion is made of a halide oran oxide as a parent material, and comprises a rare earth element whichcan emit the up-conversion emission. When a halide is used as a parentmaterial, it is advantageous because an excellent light emittingefficiency is obtained. When an oxide is used as a parent material, itis advantageous because resistance to environmental conditions, e.g.,waterproof, is high and thus stable light emitting can be obtained.

In the present invention, it is preferable that the rare earth elementis at least one of rare earth element selected from the group consistingof erbium (Er), holmium (Ho), praseodymium (Pr), thulium (Tm), neodymium(Nd), gadolinium (Gd), europium (Eu), ytterbium (Yb), samarium (Sm) andcerium (Ce). As rare earth elements capable of emitting up-conversionemission, these are preferable.

In the above mentioned invention, the fine particle containing a rareearth element can be formed from a fine particle of 1 to 100 nm averageparticle diameter of the oxide doped whit the rare earth element. Theoxide is preferably at least one of oxide selected form the groupconsisting of yttrium oxide, gadolinium oxide, lutetium oxide, lanthanumoxide and scandium oxide. This is because as the oxides doped with therare earth element, these are preferable.

A fluorescent body is used, in most cases, in powdery form. The averageparticle diameter of the fluorescent body is in a range of approximately3 to 12 μm. When the particle diameter is reduced, the light emittingefficiency starts to decrease below a certain particle diameter(approximately 1 to 2 μm, although it depends on the type of thematerial). It is assumed that it is due to the light emitting efficiencyat a surface layer of crystal is low.

In 1994, it was reported that a high light emitting efficiency isobtained in fluorescent particles having particle diameter of a few toseveral dozrn nm, and attracted attention (“Optical Properties ofManganese-Doped Nanocrystals of ZnS” APPLIED PHYSICS LETTERS, VOLUME 72,NUMBER 317, JANUARY, 1994). This phenomenon was explained on the basisof and confining emit of excitons.

Therefore, by setting the particle diameter of the fine particlescontaining a rare earth element at approximately 100 nm or less, thelight emitting efficiency of up-conversion emission by the fineparticles containing a rare earth element is likely to be furtherenhanced.

The presence of fine particles containing a rare earth element, whoseparticle diameter is approximately 100 nm or less, cannot be visuallyconfirmed, unless the fine particles containing a rare earth element isemitting light. Therefore, they are suitable as a marker for afluorescent test, such as a marker for telling the real from the falseof a holographic element.

In the present invention, erbium and ytterbium can be used as theabove-mentioned rare earth element. By regulating the amounts to beadded of these two types of rare earth elements, the visually-perceivedcolor of the light emitted from the fine particles containing a rareearth element, as a result of the up-conversion, can be controlledwithin a wavelength range from green to red.

Furthermore, erbium may be used as the above-mentioned rare earthelement. By doping yttrium oxide with erbium, the fine particlescontaining a rare earth element which emits green fluorescence byup-conversion can be obtained.

Alternatively, thulium and ytterbium can be used as the above-mentionedrare earth element. When thulium and ytterbium are added, sensitive tolight in the near infrared region can be obtained, whereby it becomespossible to induce blue light by excitation light in the near infraredregion.

In the above invention, the aforementioned fine particles containing arare earth element can be manufactured by a method for manufacturing afine particle containing a rare earth element comprising: a firstprocess of doping the rare earth element to at least one kind of basiccarbonate selected from the group consisting of basic yttrium carbonate,basic gadolinium carbonate, basic lutetium carbonate, basic lanthanumcarbonate and basic scandium carbonate; and a second process ofcalcination of the basic carbonate doped with the rare earth element.

Moreover, it is preferable that, the basic carbonate doped with the rareearth element is obtained by a liquid phase reaction, in the firstprocess. When the basic carbonate is obtained by a liquid phasereaction, fine particles containing a rare earth element having a smallparticle diameter can be obtained more easily.

In the above mentioned invention, it is preferable that, at least onekind of nitrate selected from the group consisting of yttrium nitrate,gadolinium nitrate, lutetium nitrate, lanthanum nitrate and scandiumnitrate; a nitrate of the rare earth element to be doped; and sodiumcarbonate, are reacted in the first process. When these are reacted in aliquid phase, the aforementioned basic carbonate can be obtained moreeasily.

Further, it is preferable that the basic carbonate doped with the rareearth element is quenched after calcination by a rapid heating, in thesecond process. By calcination by rapid heating and then rapidlycooling, the fine particles containing a rare earth element, whoseparticle diameter is small, can be obtained easily.

In the above mentioned invention, it is preferable that the rare earthelement is selected depending on the wavelength of the desiredfluorescence. As described above, the wavelength of the up-conversionemission from the fine particles containing a rare earth element changesdepending on the type of the rare earth element which is to be doped onan oxide. Accordingly, it is advantageous, in practical terms, to obtainthe up-conversion emission whose wavelength is suitable for theapplication of the fine particles containing a rare earth element.

The present invention also provides a fluorescent probe comprising: theabove mentioned fine particle containing a rare earth element; and aspecific binding substance which binds to the fine particle containing arare earth element. Such a fluorescent probe has the advantage of thefine particles containing a rare earth element, i.e., it is notnecessary to use UV light or blue light as excitation light. Thus, itdose not damage the biopolymer as the subject to be analyzed, does notcause problems such as lack of stability in storage like an organicfluorescent body, and has an advantage of high light emittingefficiency.

In the present invention, it is preferable that the above mentionedspecific binding substance is any one of, polynucleotide, hormone,protein, antigen, antibody, peptide, cell or tissue. The presentinvention is unique in that red light or infrared light which is lesslikely to damage biopolymer is used as excitation light. Particularly inthese above mentioned substances, damage by the excitation light islikely to be a problem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory diagram for explaining up-conversion emission.

FIG. 2 is an explanatory diagram for explaining two-photon lightemitting.

FIG. 3 is a schematic diagram for explaining an usage embodiment of thepresent invention in an antigen-antibody reaction.

FIG. 4 is a graph showing light emitting spectrum resulting fromexcitation by semiconductor laser (980 nm) of Y₂O₃: Yb, Er fineparticles.

FIG. 5 is a graph showing light emitting spectrum resulting fromexcitation by semiconductor laser (980 nm) of Y₂O₃: Er fine particles.

FIG. 6 is a graph showing light emitting spectrum resulting fromexcitation by semiconductor laser (980 nm) of Y₂O₃: Yb, Tm fineparticles.

FIG. 7 is a graph showing light emitting spectrum resulting fromexcitation by semiconductor laser (980 nm) of the fine particlescontaining a rare earth element, which are obtained by fixing the amountof erbium to be added to yttrium oxide at 1 atom %, while changing theamount of ytterbium to be added to 0 atom %, 1 atom %, 3 atom %, 5 atom% and 20 atom %, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the fine particles containing a rare earth element and afluorescent probe using the same will be described in detail.

A. Fine Particles Containing a Rare Earth Element

The fine particles containing a rare earth element of the presentinvention are characterized in that they are excited by light having awavelength in a range of 500 to 2000 nm and thereby emit up-conversionemission.

First, the up-conversion emission utilized in the present invention willbe described with reference to FIG. 1. In FIG. 1, one example, which isa system using two types of rare earth elements, i.e., ytterbium (Yb)and erbium (Er) as the rare earth element, and in which infrared lightof 1000 nm has been irradiated as excitation light, is shown. As shownin FIG. 1A, ytterbium (Yb³⁺) is first excited by excitation light of1000 nm, thereby being shifted from ²F_(7/2) level to ²F_(5/2) levelwhich is higher in energy level. This energy then pushes up, due to theenergy transfer 1, the energy level of erbium (Er³⁺) from ⁴I_(15/2)level to ⁴I_(ll/2) level. Thereafter, as shown in FIG. 1B, ytterbium(Yb³⁺) is excited by excitation light of 1000 nm and this energy pushesup, due to the energy shift 2, the energy level of erbium (Er³⁺) from⁴I_(11/2) level to ⁴F_(11/2) level. Then, as shown in FIG. 1C, the abovementioned excited erbium (Er³⁺) emits light of 550 nm when returning tothe ground state.

Thus, when a substance excited by light of 1000 nm emits light of 550nm, having higher energy, that is, in terms of wavelength, when anexcited substance emits light having higher energy than the excitationlight, such light emitting is referred to as “up-conversion emission”.

The Si nanoparticles, described in the aforementioned Background Art,which cause two-photon excitation is excited only when two photons areabsorbed at the same time, as shown in FIG. 2, and is different inprinciple from the above-mentioned up-conversion emission. Thistwo-photon excitation has poor light emitting efficiency because twophotons must exist simultaneously. On the contrary, the up-conversionemission is free of such requirements and has extremely high lightemitting efficiency, as compared with the Si nanoparticles which causetwo-photon excitation.

In the present invention, since a rare earth element which emits theaforementioned up-conversion emission is employed, there is no need forexcitation by light having high energy such as UV light. That is, thewavelength of light when emitting light is preferably, in general, thevisible light so that analysis or detection thereof is easy. Therefore,for up-conversion emission, light having a longer wavelength thanvisible light is used as excitation light. As a result, UV light or bluelight which is likely to damage biopolymer is prevented from being usedas excitation light. Further, as the wavelength of the excitation lightand the wavelength of the emitted light are hardly overlapped, analysisor detection is significantly facilitated.

As described above, as the fine particles containing a rare earthelement of the present invention employ a rare earth element which canemit up-conversion emission, the excitation light does not damage thesubject to be analyzed and thus an accurate analysis can be done.Further, the light emitting efficiency is much higher than thetwo-photon excitation and the stability during storage is excellent, ascompared with the case of using the organic fluorescent body. Thus, astable and highly precise analysis can be done.

The fine particles containing a rare earth element of the presentinvention, which contain a rare earth element capable of emitting suchup-conversion emission, for example, may be constituted from a coreportion containing the rare earth element capable of emitting theup-conversion emission, and a functional shell portion having a functionof being bound to a specific binding substance and preventingagglutination (hereinafter, the fine particles containing a rare earthelement of this type will be referred to as “first fine particlescontaining a rare earth element”). Alternatively, they may beconstituted from an oxide doped with a rare earth metal having averageparticle diameter of 1 to 100 nm and (hereinafter, such fine particlescontaining a rare earth element will be referred to as “second fineparticles containing a rare earth element”).

Hereinafter, the fine particles containing a rare earth element of thepresent invention will be described, first on the first fine particlescontaining a rare earth element and then on the second fine particlescontaining a rare earth element.

(I) First Fine Particles Containing a Rare Earth Element

The diameter of the first fine particles containing a rare earth elementas a whole is preferably in a range of 1 to 500 nm, and especiallypreferably in a range of 1 to 100 nm. A surface of such first fineparticles containing a rare earth element, i.e., the functional shellportion, is preferably provided with a functional group. By this,binding with a specific binding substance is facilitated.

The first fine particles containing a rare earth element as describedabove, of the present invention, will be described in detail, first onthe core portion and then on the functional shell portion.

1. Core Portion

The core portion of the first fine particles containing a rare earthelement of the present invention contains a rare earth element. First,such rare earth element will be described, followed by the descriptionof the core portion containing the rare earth element.

(1) Rare Earth Element

The rare earth element used in the present invention is not particularlylimited as long as it is a rare earth element, which is excited by lighthaving a wavelength in a predetermined range, as describe above, and canthereby emit up-conversion emission.

With regards to the range of the wavelength of the excitation light, thewavelength needs to be at least in a range of 500 to 2000 nm, preferablyin a range of 700 to 2000 nm, and particularly preferably in a range of800 to 1600 nm, so that the excitation light does not damage biopolymer.

Examples of such a rare earth element generally include a rare earthelement which can be a trivalent ion. Specifically, rare earth elementssuch as erbium (Er), holmium (Ho), praseodymium (Pr), thulium (Tm),neodymium (Nd), gadolinium (Gd), europium (Eu), ytterbium (Yb), samarium(Sm) and cerium (Ce) can be suitably used.

In the present invention, the rare earth element capable ofup-conversion emission as described above may be used either as a singletype of element or as a combination of two or more types of elements ata time. The mechanism of up-conversion emission when a single type of arare earth element is used, explained using Er³⁺ dope material as anexample is as follows: when light of 970 nm or 1500 nm is irradiated asexcitation light, followed by the up-conversion process, visible lightof such as 410 nm (²H_(9/2)-⁴I_(15/2)), 550 nm (⁴S_(3/2)-⁴I_(15/2)), 660nm (⁴F_(9/2)-⁴I_(15/2)) is emitted in accordance with the energy levelof Er³⁺ ion.

(2) Core Portion

The the core portion containing a rare earth element is not particularlylimited as long as the core portion contains the rare earth elementtherein in a state in which up-conversion emission can be emitted, andthe core portion may be an organic matter, for example, such that formedin a state of comprising the rare earth element in a complex, adendrimer, or the like. However, the core portion formed by mixing theaforementioned rare earth elements in an inorganic parent material isgenerally preferable because, it is easy to make the rare earth elementcontained in a state that can emit light.

As the inorganic parent material, a material which has transparency toexcitation light is preferable in terms of light emitting efficiency.Specifically, a halide such as a fluoride and a chloride, an oxide, asulfide and the like can be suitably used.

In the present invention, a halide is suitably used in terms of lightemitting efficiency. Specific examples of such a halide include bariumchloride (BaCl₂), lead chloride (PbCl₂), lead fluoride (PbF₂), cadmiumfluoride (CdF₂), lanthanum fluoride (LaF₃), yttrium fluoride (YF₃) andthe like. Among these examples, barium chloride (BaCl₂), lead chloride(PbCl₂) and yttrium fluoride (YF₃) are preferable.

As an example of the parent material with high environmental resistancesuch as being stable to moisture, an oxide can be raised. Specificexamples of such an oxide include yttrium oxide (Y₂O₃), aluminum oxide(Al₂O₃), silicon oxide (SiO₂), tantalum oxide (Ta₂O₅), and the like.Among these examples, yttrium oxide (Y₂O₃) is preferable.

When a halide is used as the parent material, it is preferable that aprotection layer is formed around. A halide is generally unstable towater and the like, and if halide particles are used as they are,analysis may not be done accurately. In such cases, it is preferablethat the core portion is formed as a composite core portion, whichcomprises a coating material having waterproof ability or the likeformed around fine particles made of a halide as the parent material. Asthe coating material in such cases, the oxide as described above can besuitably used.

Examples of a method of introducing a rare earth element to a parentmaterial include: in a case of a halide such as barium chloride (BaCl₂),methods described in JP-A 9-208947 Laid-Open or a reference (“Efficient1.5 mm to Visible Upconversion in Er³⁺ Doped Halide Phoshors” JunichiOhwaki et al., p. 1334-1337, JAPANESE JOURNAL OF APPLIED PHYSICS, Vol.31 part 2 No. 3A, 1 Mar. 1994). In a case of an oxide, methods describedin JP-A 7-3261 Laid-Open or references (“Green Up conversionFluorescence in Er³⁺ Doped Ta₂O₅ Heated Gel” Kazuo Kojima et al., Vol.67(23), 4 Dec. 1995; and “Relationship Between Optical Properties andCrystallinity of Nanometer Y₂O₃: Eu Phoshor” APPLIED PHYSICS LETTERS,Vol. 76, No. 12, p. 1549-1551, 20 Mar. 2000).

In the present invention, the amount of the rare earth element to beintroduced in the above mentioned parent material changes significantlydepending on the type of the rare earth element, the type of the parentmaterial, or the desired degree of intensity of light emitting.Therefore, the amount is set in an appropriate manner, in considerationof the various conditions.

The average particle diameter of the core portion is preferably in arange of 1 to 100 nm and more preferably in a range of 1 to 50 nm.Synthesis of fine particles with average particle diameter of the coreportion of less than 1 nm would be extremely difficult and thus notpreferable. On the other hand, fine particles with average particlediameter of the core portion exceeding 100 nm tend to disturb thereaction of the subject to be labeled, the specific binding substance,thereby deteriorating the precision of data, thus not preferable.

The color of the emitting light of the up-conversion emission of therare earth elements differs depending on the composition. Therefore, byutilizing this feature, by labeling plural types of specific bindingsubstances having different specificity, with different color lightemitting fine particles containing a rare earth element respectively,and conducting the same measurement, it is possible to detect differenttypes of testing substances simultaneously.

2. Functional Shell Portion

The first fine particles containing a rare earth element of the presentinvention has the functional shell portion formed around the coreportion. The functional shell portion is required not to be agglutinatedin a liquid having a relatively high salt concentration such as bodyfluid, and not to perform an on-specific reaction in a sample liquid.

The specific characteristics which are required to such functional shellportion, first, having binding ability to a specific substance can bepointed out. That is, it is preferable that the functional shell portionhas a specific binding substance binding site, which is the site bindsto a specific binding substance and it is preferable that the functionalshell portion is formed of a material comprising such sites.

Such a specific binding substance binding site as described above is notparticularly limited as long as the site provide physical bindingcapacity or chemical binding capacity to a surface of the shell portion.Specific examples thereof include a site with ion dissociation ability,a site with ion coordination ability, a site with a function to bind toa metal, a site with condensation reactivity, a site with additionreactivity, a site with substitution reactivity, a site with hydrogenbonding ability, and a site capable of effecting a specific interaction.Among these examples, the site is preferably at least one of a site withcondensation reactivity, a site with addition reactivity, a site withsubstitution reactivity, and a site capable of effecting a specificinteraction.

Specific examples of such a preferable binding site include carboxylgroup, amino group, hydroxyl group, aldehyde group (—CHO), vinyl group(CH₂═CH—), acryloyl group, methacryloyl group, epoxy group, acetal group((CH₃CH₂O)₂CH—), an imide site and a biotin site.

Next, as characteristic which is required to the functional shellportion is that the functional shell portion is capable of preventingthe first fine particles containing a rare earth element fromagglutination. Accordingly, it is preferable that the functional shellportion has an agglutinate preventing site which prevents the first fineparticles containing a rare earth element from agglutination, and isformed of a material comprising such sites.

The agglutinate preventing site is preferably at least one of a site ofa site having hydrogen bonding or a site with hydrating ability.Specific examples thereof include ethylene oxide site (—(CH₂CH₂O)_(n)—)(n is an integer in a range of 2 to 10,000), hydroxyl group (—OH), amidesite (—CONH—), phosphate ester site (—PO(OR)_(n)(OH)_(3-n), (n is 1, 2or 3, R represents a hydrocarbon-containing group having 2 or morecarbon atoms)) and a betaine site. It is especially preferable that theagglutinate preventing site is one of an ethylene oxide site(—(CH₂CH₂O)_(n)—) (n is an integer in a range of 2 to 10,000) typicallyrepresented by polyethylene glycol, or betaine site, because they excelin function to form a stable hydration layer on the surface of the fineparticles and excel in function to prevent agglutination or non-specificadsorption.

Other preferable examples of the site with agglutinate preventingfunction include an ion-dissociation site which effects electrostaticrepulsion. Specific examples thereof include carboxyl group, sulphonicgroup, amino group, imino group, betaine group. A polymer comprisingthese groups is especially preferable, because such a polymer excels infunctions to form a stable electrostatic repulsion layer on the surfaceof the fine particles.

Further, in the present invention, it is preferable that the functionalshell portion comprises a core portion binding site, which binds to thecore portion, therefore, it is preferable that it is formed of amaterial comprising such sites. Such site may include at least one of asite with a function to bind to a metal, a site with condensationreactivity, a site with addition reactivity, and a site withsubstitution reactivity.

Specific examples include methoxysilyl group (—Si (OCH₃)_(n)Y_(n−1) (nis 1, 2 or 3, Y represents methyl or ethyl)), ethoxysilyl group(—Si(OCH₂CH₃)_(n)Y_(n−1) (n is 1, 2 or 3, Y represents methyl orethyl)), propoxysilyl group (—Si (OC₃H₇)_(n)Y_(n−1) (n is 1, 2 or 3, Yrepresents methyl or ethyl)), chlorosilyl group (—SiCl_(n)Y_(n−1) (n is1, 2 or 3, Y represents methyl or ethyl)), vinyl group, acryloyl group,methacryloyl group and the like.

Further, it is preferable that the functional shell portion is formed ofa material with a function to prevent deterioration of light emittingefficiency of the first fine particles containing a rare earth element,due to non-radiation, a chemical reaction, or other causes. Also, amaterial which firmly coordinates or binds to the surface of the coreportion of the first fine particles containing a rare earth element ispreferable. It is especially preferable that such a material ispolymerizable. That is, materials having a site with ion coordinationability, a site with a function to bind to a metal, a site withcondensation reactivity, a site with addition reactivity, or a site withsubstitution reactivity are preferable. Specific examples thereofinclude materials comprising carboxyl group, amino group, imino group,thiol group, Schiff base site (—CH═N—), methoxysilyl group(—Si(OCH₃)_(n)Y_(n−1) (n is 1, 2 or 3, Y represents methyl or ethyl)),ethoxysilyl group (—Si(OCH₂CH₃)_(n)Y_(n−1) (n is 1, 2 or 3, Y representsmethyl or ethyl)), propoxysilyl group (—Si (OC₃H₇)_(n)Y_(n−1) (n is 1, 2or 3, Y represents methyl or ethyl)), chlorosilyl group(—SiCl_(n)Y_(n−1) (n is 1, 2 or 3, Y represents methyl or ethyl)), vinylgroup, acryloyl group, methacryloyl group and the like.

As described above, the functional shell portion is required to havevarious functions. Therefore, as materials forming such functional shellportion, a material having the above mentioned desired functions as acomplex is preferable. Chemical formulae of specific examples of such apreferable material are shown below.

3. Method for Manufacturing the First Fine Particles Containing a RareEarth Element

Examples of a method for manufacturing the core portion of the firstfine particles containing a rare earth element include: theevaporation-in-gas method including the high frequency plasma method;spattering method; glass crystallization method; chemical precipitationmethod; the reverse micelle method; the sol-gel method and equivalentmethods thereof; precipitation method including hydrothermal synthesismethod and coprecipitation method; and spraying method.

Preferable examples of a method of forming the functional shell portionon the surface of the core portion of the first fine particlescontaining a rare earth element include: a method of forming covalentbond of the functional group of the material constituting the functionalshell portion, with the functional group of the core portion surface, bya condensation or addition reaction; a method of synthesizing thefunctional shell portion in the presence of the core portion by thesol-gel method or an equivalent method thereof; and a method of makingthe core portion adsorb a precursor of the functional shell portion andthen polymerizing. When the core portion is synthesized by chemicalprecipitation method, the reverse micelle method, the sol-gel method oran equivalent method thereof, it is possible to utilize a surfactant ora protecting agent used at the time, as the shell portion or theprecursor of the shell portion.

(II) Second Fine Particles Containing a Rare Earth Element

1. Second Fine Particles Containing a Rare Earth Element

The second fine particles containing a rare earth element are fineparticles having average particle diameter of 1 to 100 nm and comprisingan oxide as a parent material doped with a rare earth element. Thesecond fine particles containing a rare earth element correspond to onepreferable embodiment of the core portion of the above mentioned firstfine particles containing a rare earth element. However, the second fineparticles containing a rare earth element are different from the firstfine particles containing a rare earth element in that the functionalshell portion is not an essential component.

The oxide used as the parent material may be selected from the groupcomprising yttrium oxide, gadolinium oxide, lutetium oxide, lanthanumoxide and scandium oxide. The number of oxide may be of either a singletype or two or more types of the aforementioned examples.

The rare earth element to be doped with on the parent material may beeither of a single type or two or more types of the rare earth elementsaccording to the wavelength of the desired up-conversion emission.

For example, in a case in which yttrium oxide is doped with erbium, thesecond fine particles containing a rare earth element, which emit greenlight as a result of up-conversion caused by excitation by light havinga wavelength of 980 nm, can be obtained.

Alternatively, in a case in which yttrium oxide is doped with erbium andytterbium, the second fine particles containing a rare earth element,which emits light of a predetermined color within the wavelength rangeof green to red, in a visually recognizable manner as a result ofup-conversion caused by excitation by light having a wavelength of 980nm, can be obtained. For example, when erbium is added by the amount of1 mol % and ytterbium is added by the amount of 20 mol %, the secondfine particles containing a rare earth element, which emits red light ina visually recognizable manner by excitation by light having awavelength of 980 nm, can be obtained.

FIG. 7 shows spectra of fluorescence by up-conversion, obtained from thesecond fine particles containing a rare earth element obtained by fixingthe content of erbium added to yttrium oxide at 1 atom % while changingthe content of ytterbium added at 0 atom %, 1 atom %, 3 atom %, 5 atom %and 20 atom %, respectively. In FIG. 7, spectra at 0 atom %, 1 atom %, 3atom %, 5 atom % and 20 atom % of the added yttrium amount are shown asspectrum (i) to (iv), respectively, in this order.

As is obvious from FIG. 7, the color of light which is visuallyrecognized when the second particles containing a rare earth element isexcited changes within the wavelength range of green to red, inaccordance with the ratio of the adding amount of erbium and ytterbium.

In a case of yttrium oxide is doped with thulium and ytterbium, as thesensitivity to light in the near infrared region is increased byytterbium, light emitting of blue light due to thulium can be obtainedwith higher intensity when excited by light having a wavelength of 980nm.

The amount of a rare earth element to be added to ytterbium oxide at thetime maybe appropriately selected within in a certain range whichconcentration quenching dose not occur. For example, when erbium,ytterbium, or thulium are added to yttrium oxide, the added amount oferbium, ytterbium and thulium maybe selected, respectively, in anappropriate manner within a range of 0 to 50 mol %.

2. Method for Manufacturing the Second Fine Particles Containing a RareEarth Element

The aforementioned second fine particles containing a rare earth elementcan be obtained, for example, by: preparing a basic carbonate doped witha desired rare earth element; calcination of the basic carbonate; andthe then optionally adjusting the particle diameter of the particles toa predetermined size.

The above-mentioned basic carbonate may be selected from the groupcomprising basic yttrium carbonate, basic gadolinium carbonate, basiclutetium carbonate, basic lanthanum carbonate and basic scandiumcarbonate. The number of the basic carbonate may be of either a singletype or two or more types of the aforementioned examples.

The type of the rare earth element doped on the basic carbonate may beappropriately selected, as describe above, in accordance with the targetwavelength of fluorescence to be emitted from the second fine particlescontaining a rare earth element as a result of up-conversion.

In order to obtain the second fine particles containing a rare earthelement having average particle diameter of 1 to 100 nm, it ispreferable that the basic carbonate doped with the desired rare earthelement is obtained by a liquid phase reaction. Such a basic carbonatecan be obtained by reacting a nitrate of a metal which is a componentelement of the basic carbonate to be doped with a rare earth element,with a nitrate of the rare earth element used to bedoped, and sodiumcarbonate.

When a basic carbonate doped with the desired rare earth element iscalcined, it is preferable that the calcination is carried out by rapidheating, followed by rapid cooling. By heating rapidly and then coolingrapidly, growth of particles is prevented and the average particlediameter can be made no larger than 100 nm.

In the present invention, “rapid heating” represents putting in an ovenset in a range of 300 to 1700° C., preferably in a range of 500 to 1100°C. for at least 10 minutes, preferably for 30 to 180 minutes. “Rapidcooling” represents taking out of the above mentioned oven and leavingat a temperature condition which is at least 200° C., preferably 500 to1100° C. lower than the temperature inside the oven.

By going through each of the aforementioned processes, the second fineparticles containing a rare earth element as described above can beobtained.

The aforementioned second fine particles containing a rare earth elementmay be used as the core portion of the above mentioned first fineparticles containing a rare earth element.

B. Fluorescent Probe

The fluorescent probe of the present invention comprising: the abovementioned first or second fine particles containing a rare earthelement; and a specific binding substance which binds to the abovementioned first or second fine particles containing a rare earthelement.

1. Specific Binding Substance

In the present invention, fluorescent probes having various functionscan be produced by labeling a known or unknown specific bindingsubstance with the aforementioned first or second fine particlescontaining a rare earth element. For example, by labeling a knownantibody, a fluorescent probe, for analyzing the position of an antigenin a sample to be tested, can be produced. Or, by labeling anunidentified DNA (a sample to be tested), a fluorescent probe, fordetermining the base sequence of the sample by using DNA chips, can beproduced.

Examples of the specific binding substance as described above includesynthetic nucleic acid such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), peptide nucleic acid (PNA); hormone, protein, peptide, cell,tissue, antigen, antibody, receptor, hapten, enzyme, nucleic acid,medical agent, chemical substances, polymer, pathogen, toxin, adeninederivative, guanine derivative, cytosine derivative, thymine derivative,urasil derivative and the like.

In the present invention, synthetic nucleic acid such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA) and peptide nucleicacid (PNA), hormone, protein, antigen, antibody, peptide and cell, ofthe aforementioned examples, are preferably used as the specific bindingsubstance. As described above, the key feature of the present inventionlies in that excitation light does not damage biopolymer. Since even avery slight damage on DNA, RNA, synthetic nucleic acid, hormone,protein, antigen, antibody, peptide and cell by excitation could have asignificant impact on the result of the analysis, the present inventionis most advantageously applied to such biopolymer.

2. Labeling of Specific Binding Substance by Fine Particles ContainingRare Earth Element

In the present invention, a fluorescent probe is produced by labelingthe above-mentioned specific binding substance by binding with the abovementioned first or second fine particles containing a rare earthelement.

The manner of binding the fine particles containing a rare earth elementand the specific binding substance is not particularly limited, andexamples of thereof include: physical bonding such as ionic bonding,coordinate bonding and hydrogen bonding; chemical bonding, i.e.,covalent bonding; and bonding by a specific interaction. In order toimprove the precision in analysis, it is preferable that the specificbinding substance is firmly bound to the fine particles containing arare earth element. Therefore, it is preferable that they are bonded bycovalent bond or by a specific interaction.

3. Analyzing or Detecting Method

Examples of the analyzing or detecting method using the fluorescentprobe of the present invention include a method using anantigen-antibody reaction, as described below.

That is, when an antibody is reacted with a substance (antigen) on orinside a cell, the antibody is firmly bound to the antigen as a resultof an antigen-antibody reaction. Accordingly, the location of theantigen can be identified based on the binding site. However, ingeneral, the antibody molecule itself can not be observed by amicroscope. Thus, it is necessary to label the antibody to be used forantigen detection, with an observable marker in advance. Anantigen-antibody reaction is a reversible bonding reaction, as shown,for example, in FIG. 3. The reaction is very specific and the antibodyis not reacted with any substance other than the corresponding antigensubstance. The upper part of the figure shows that, when antibody isreacted to the tissue sample, the antibody is reacted with and bound toonly to antigen A. However, in this state, the location of the antigencannot be identified because the antibody itself cannot be dyed out.Therefore, as the case shown in the lower part of the figure, when avisually observable marker is bound to the antibody in advance andreacted with the antigen in the tissue, the binding similar to thatshown in the upper part of the figure occurs. In this case, the locationwhere the antigen-antibody reaction occurred is indicated by the marker,whereby the site where the antigen is present can be identified. What isimportant about the labeled antibody is that the ability to be bound tothe antigen is not lost by labeling, does not have any non-specificaffinity with other substances, and the activity of the marker issufficiently maintained.

The fluorescent antibody method (fluorescent-labeled antibody method) asdescribed above, is one of the immunohistochemical methods, in which afluorescent colorant is used as a marker of antibody and the location ofantigen is determined by observing the fluorescence emitted as a resultof excitation under a fluorescence microscope. This method was first tobe established among the immunohistochemical methods, Coons et al.started the development thereof in the early 1940's, and substantiallyestablished in 1950's. Thereafter, due to the improvement of thelabeling method by Riggs et al. and introduction of the method ofpurifying labeled antibody by McDvitt et al., the area to which thismethod is applicable has been widened, and now, this method is one ofthe most common methods in immunohistochemistry.

The present invention is applicable to the aforementioned method, byemploying the first or second fine particles containing a rare earthelement, comprising the rare earth element, as the marker, and by makingthe antibody as the specific binding substance.

As above, by binding a specific binding substance, which specificallybinds to another biopolymer, to the aforementioned fine particlescontaining a rare earth element and thus labeling, various types ofanalysis and detection are possible.

Examples of such specific binding include: combination of a nucleic acid(for example, oligonucleotide or polynucleotide) and another nucleicacid complementary thereto; combination of antigen and antibody (orantibody fragment) as described above; combination of a receptor andligand thereof (for example, hormone, cytokinin, neurotransmitter orlectin); combination of an enzyme and ligand thereof (for example,substrate analog of the enzyme, coenzyme, a regulating factor, or aninhibitor); combination of enzyme analog and a substrate of enzyme whichis the base of the enzyme analog; and combination of lectin andsaccharide. Note that the “enzyme analog” refers to a substance whichhas significantly high specific affinity to the substrate of thecorresponding original enzyme, but exhibits no catalytic activity.Further, the each compound in each of the combinations described above,respectively, either one can be “the specific binding substance”, andthe other can be the substance to be analyzed or detected. For example,in “the combination of antigen and antibody”, when the antigen serves as“the substance to be analyzed or detected”, the antibody can be “thespecific binding substance”. Conversely, when the antibody serves as“the substance to be analyzed or detected”, the antigen can be “thespecific binding substance”.

For example, in a case in which the fluorescent probe of the presentinvention is applied to a nucleic acid hybridization assay, anoligonucleotide or a polynucleotide, capable of complementarily beingbound to a nucleic acid (for example, an oligonucleotide or apolynucleotide) which is a substance to be analyzed or detected, can beused as the above mentioned specific binding substance.

Here, in the “oligonucleotide” or in the “polynucleotide”,deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and peptide nucleicacid (PNA) are comprised. “PNA” refers to an artificially synthesizednucleic acid obtained by converting the phosphodiester bond of DNA intoa peptide bond. The chain length of the oligonucleotide orpolynucleotide bound to the aforementioned insoluble particles maybeselected appropriately, based on the object of the analysis, forexample, it may be determined based on the chain length of acomplementary sequence of DNA, RNA or PNA to be captured.

The synthesis of oligonucleotide used as the above mentioned specificbinding substance can generally be carried out by using an automaticsynthesizing device. Moreover, in can be carried out by a conventionalgenetic engineering technique such as PCR (polymerase chain reaction).

When the fluorescent probe of the present invention is applied to animmunological assay, an antigen (including hapten) or an antibody, whichbinds specifically to a substance to be analyzed or detected, can beused as the specific binding substance. In this case, the substance tobe analyzed or detected is not particularly limited as long as thesubstance is a component generally contained in the sample to be testedand also a substance which can be immunologically detected. Examples ofsuch a substance include proteins of various types, polysaccharides,lipid, biomass, various types of environmental substance and the like.More specific examples thereof include immunoglobulin (for example, IgG,IgM or IgA), infection disease-associated marker (for example, HBsantigen, HBs antibody, HIV-1 antibody, HIV-2 antibody, HTLV-1 antibody,or treponemal antibody), tumor-associated antigen (for example, AFP,CRP, or CEA), coagulation/fibrinogenolysis marker (for example,plasminogen, anti-thrombin-III, D-dimer, TAT, or PPI), antiepileptics(for example, hormone), various types of medical agents (for example,digoxin), biomass (for example, O-157, or Salmonella) or endotoxin orexotoxin thereof, microorganisms, enzymes, agricultural chemicalsresidue, and environmental endocrine disrupters. Either polychlonalantibody or monoclonal antibody, which can be obtained by theconventional method, can be used as the antibody used as the specificbinding substance. Further, as the antibody, antibody which has beensubjected to a protein (for example, enzyme such as pepsin or papain)treatment, for example, antibody fragments such as Fab, Fab′, F(ab′)₂,or Fv, may be used.

As described above, the fluorescent probe of the present invention canbe used for detection or analysis of biopolymer of various types.Further, the fluorescent probe of the present invention has an advantagethat, when excitation light is irradiated, no damage is done tobiopolymer to be detected or analyzed.

The present invention is not limited to the aforementioned embodiment.The aforementioned embodiment is an example, and whatever issubstantially equivalent in structure and has similar effect to thetechnological thoughts described in the claims of the present invention,is included to the technological scope of the present invention.

EXAMPLES

Hereinafter, the present invention will be described in detail withreference to the following examples.

1. Production of Core Portion, or the Second Fine Particles Containing aRare Earth Element

First, a method for producing a core portion containing a rare earthelement characterized in emitting the up-conversion emission will bedescribed.

1-1. Y₂O₃: Yb, Er Fine Particles

Y₂O₃: Yb, Er fine particles were produced by preparing a precursor dopedwith Yb and Er, by a liquid phase reaction, and then by calcination ofthe precursor. The manufacturing process is as follows.

First, 0.0158 mol of yttrium nitrate, 0.004 mol of ytterbium nitrate and0.0002 mol of erbium nitrate were dissolved in distilled water such thatthe volume of the mixture was 100 ml, thereby preparing a Y, Yb, Erion-mixture solution. 100 ml of sodium carbonate aqueous solution (0.3mol/l) was added to the above mentioned Y, Yb, Er ion-mixture solution,and the mixture was stirred for 2 hours.

Next, the mixture was subjected to centrifugation three times, each at3000 rpm for 30 minutes, by using a centrifuge. Thereafter, theprecipitation was dried in vacuum at 45° C. for hours, whereby basicyttrium carbonate doped with Yb and Er, as a precursor, was obtained.

The obtained precursor was put in an electric oven and rapidly heated,after being maintained in the oven at 900° C. for 30 minutes in the airatmosphere, the precursor was taken out of the oven and rapidly cooled.As a result, Y₂O₃: Yb, Er fine particles were synthesized. From themeasurement results of SEM and XRD, it was confirmed that the averageparticle diameter was approximately 30 nm.

The light emitting spectrum of the resulting Y₂O₃: Yb, Er fine particlesof light excitation by a semiconductor laser (980 nm) is shown in FIG.4. Emission of green color of Er³⁺ is observed in the vicinity of 550 nmand emission of red color of Yb 3+is observed in a wavelength range of650 to 680 nm.

It should be noted that FIG. 4 shows light emitting spectrum of Y₂O₃:Yb, Er fine particles to which erbium was added by the amount of 1 mol %and ytterbium was added by the amount of 20 mol %.

1-2. Y₂O₃: Er Fine Particles

Y₂O₃: Er fine particles were produced by preparing a precursor dopedwith Er, by a liquid phase reaction, and then by calcination of theprecursor. The manufacturing process is as follows.

First, 0.0198 mol of yttrium nitrate and 0.0002 mol of erbium nitratewere dissolved in distilled water such that the volume of the mixturewas 100 ml, thereby preparing a Y, Er ion-mixture solution. 100 ml ofsodium carbonate aqueous solution (0.3 mol/l) was added to the abovementioned Y, Er ion-mixture solution, and the mixture was stirred for 2hours.

Next, the mixture was subjected to centrifugation three times, each at3000 rpm for 30 minutes, by using a centrifuge. Thereafter, theprecipitation was dried in vacuum at 45° C. for 5 hours, whereby basicyttrium carbonate doped with Er, as a precursor, was obtained.

The obtained precursor was put in an electric oven and rapidly heated,after being maintained at 900° C. for 30 minutes in the air atmosphere,the precursor was taken out of the oven and rapidly cooled. As a result,Y₂O₃: Er fine particles were synthesized. From the measurement resultsof SEM and XRD, it was confirmed that the average particle diameter wasapproximately 30 nm.

The light emitting spectrum of the resulting Y₂O₃: Er fine particles oflight excitation by a semiconductor laser (980 nm) is shown in FIG. 5.Emission of green color of Er³⁺ is observed in the vicinity of 550 nm.

It should be noted that FIG. 5 shows light emitting spectrum of Y₂O₃: Erfine particles to which erbium was added by the amount of 1 mol %.

1-3. Y₂O₃: Yb, Tm Fine Particles

Y₂O₃: Yb, Tm fine particles were produced by preparing a precursor dopedwith Yb and Tm, by a liquid phase reaction, and then by calcination ofthe precursor. The manufacturing process is as follows.

First, 0.0158 mol of yttrium nitrate, 0.004 mol of ytterbium nitrate and0.0002 mol of thulium nitrate were dissolved in distilled water suchthat the volume of the mixture was 100 ml, thereby preparing a Y, Yb, Tmion-mixture solution. 100 ml of sodium carbonate aqueous solution (0.3mol/l) was added to the above mentioned Y, Yb, Tm ion-mixture solution,and the mixture was stirred for 2 hours.

Next, the mixture was subjected to centrifugation three times, each at3000 rpm for 30 minutes, by using a centrifuge. Thereafter, theprecipitation was dried in vacuum at 45° C. for 5 hours, whereby basicyttrium carbonate doped with Yb and Tm, as a precursor, was obtained.

The obtained precursor was put in an electric oven and rapidly heated,after being maintained at 900° C. for 30 minutes in the air atmosphere,the precursor was taken out of the oven and rapidly cooled. As a result,Y₂O₃: Yb, Tm fine particles were synthesized. From the measurementresults of SEM and XRD, it was confirmed that the average particlediameter was approximately 30 nm.

The light emitting spectrum of the resulting Y₂O₃: Yb, Tm fine particlesof light excitation by a semiconductor laser (980 nm) is shown in FIG.6. Emission of blue color of Tm³⁺ is observed in the vicinity of 480 nm.

It should be noted that FIG. 6 shows light emitting spectrum of Y₂O₃:Yb, Tm fine particles to which thulium was added by the amount of 1 mol% and ytterbium was added by the amount of 20 mol %.

2. Production of the First Fine Particles Containing a Rare EarthElement

2-1. Y₂O₃: Yb, Er/APTS Fine Particles

300 mg of Y₂O₃: Yb, Er fine particles synthesized in the aforementioned1.1 was first dispersed in a small amount of dry toluene containing 30mg of a polymer dispersion stabilizer and then the mixture was dilutedwith additional dry toluene such that the total weight of the mixturewas 10 g. 0.5 g of (3-aminopropyl)trimethoxysilane (APTS) was added tothe thus obtained fine-particle-dispersed solution in nitrogenatmosphere, with vigorous stirring. The stirring was continued at theroom temperature for 15 hours.

Then, the APTS-treated fine particles were collected by using acentrifuge. Thereafter, the collected fine particles were dispersed totoluene again, centrifuged, collected, dispersed to toluene:methanol(1:1), centrifuged, collected, dispersed to methanol, centrifuged,collected, dispersed to methanol:water (1:1), centrifuged, collected,finally dispersed to water and centrifuged, whereby Y₂O₃: Yb, Er/APTScomposite fine particles were obtained.

The yield was 210 mg. The dried composite fine particles weresuccessfully dispersed to water again. This composite fine particleexhibited up-conversion emission, both in a dry state and in awater-dispersed state, having spectrum shape similar to that of FIG. 4.

2-2. Y₂O₃: Er/PEG-Biotin Fine Particles

By using 300 mg of the Y₂O₃: Er fine particles synthesized in theaforementioned 1-2, Y₂O₃: Er/APTS composite fine particles were producedin a manner similar to that of 2-1. The yield was 233 mg. 200 mg ofY₂O₃: Er/APTS composite fine particles were dispersed again to 5 g ofwater of pH 8.

A solution, prepared by adding 30 mg of Biotin-PEG-NHS (compound 16shown above, manufactured by Shearwater Co. Ltd., catalogue No.OH2ZOF02) to 5 g of water of pH 8, was added to the above dispersionliquid. The mixture was stirred for 1 hour. Thereafter, theBiotin-PEG-NHS treated composite fine particles was separated from thesupernatant liquid by centrifuge, the composite fine particles weredispersed to water again, centrifuged and collected repeated for threetimes. Thereafter, the composite fine particles were purified bydialysis, whereby the target Y₂O₃: Er/PEG-Biotin composite fineparticles were obtained.

The total weight of the obtained dispersion solution of Y₂O₃:Er/PEG-Biotin composite fine particles was 33.4 g. The yield of Y₂O₃:Er/PEG-Biotin composite fine particles, which was obtained by collectinga portion thereof and measuring a change in weight of before and afterbeing dried, was 74 mg. The composite particles exhibited up-conversionemission, both in a dry state and in a water-dispersed state, havingspectrum shape similar to that of FIG. 5.

2-3. Y₂O₃: Er/(APTS-containing Layer) Fine Particles

20 mmol of anhydrous yttrium acetate and 0.4 mmol of anhydrous erbiumacetate were dispersed to anhydrous ethanol. The obtained anhydrousethanol dispersion solution was refluxed for 3 hours and then cooled tothe room temperature. While being cooled to 0° C. and with vigorousstirring being irradiated with ultrasonic wave, an anhydrous methanolsolution of tetramethylammonium hydroxide was added thereto, wherebyY₂O₃: Er fine particles were produced. The amount of addedtetramethylammonium was approximately 22 mmol.

To this Y₂O₃: Er fine particles dispersion solutuin, an anhydroustoluene solution containing 0.01 mmol of tetraethoxysilane and 0.01 mmolof APTS was added. The mixture was allowed to be reacted in nitrogenatmosphere for further 2 hours, whereby Y₂O₃: Er/(APTS-containing layer)composite fine particles were produced. The dispersion liquid of Y₂O₃:Er/(APTS-containing layer) composite fine particles, which agglutinatewas removed, was twice subjected to the process in which the fineparticles were collected by centrifugation and dispersed to ethanol. Theobtained fine particles dispersion liquid was flowed into a dialysistube with a stirrer bar, and stirred with a magnetic stirrer, in waterof pH 3.8, for 3 days, with water constantly being replaced. Thereafter,semiconductor laser (980 nm) was irradiated to the resulting to-waterdispersion liquid of Y₂O₃: Er/(APTS-containing layer) composite fineparticles in a dark place. It was confirmed that the irradiated portionemitted green color light. The average particle diameter measured by atransmission electron microscope was approximately 11 nm.

2-4. LaF₃: Yb, Er/(APTS-containing Layer) Fine Particles

120 mmol of di-n-octadecyldithiophosphate (DOSP) and 120 mmol of sodiumfluoride were added to 4 L of an ethanol/water mixed solvent (10:1), andheated to 75° C. To this heated mixture solution, 100 ml of an aqueoussolution containing 40 mmol of lanthanum nitrate, 0.12 mmol of ytterbiumnitrate and 0.01 mmol of erbium nitrate was added dropwise and wascontinued to be heated at 75° C. for 2 hours. After the mixture solutionwas cooled to the room temperature, rough LaF₃: Yb, Er/(DOSP-containinglayer) fine particles were separated by centrifugation and the cleanedwith water and ethanol for five times each. Thereafter, the fineparticles were dispersed to dichloromethane, precipitated again byadding ethanol. The precipitation was separated by centrifugation. Thisprocess was repeated for three times, whereby LaF₃: Yb,Er/(DOSP-containing layer) fine particles were purified. The solidcontent obtained after being vacuum-dried at the room temperature for 24hours weighed 6.9 g. The obtained LaF₃: Yb, Er/(DOSP-containing layer)fine particles were dispersible to dichloromethane or toluene, but notdispersible to ethanol and water. 5 g of LaF₃: Yb, Er/(DOSP-containinglayer) fine particles was dispersed to 500 ml of anhydrous toluene innitrogen atmosphere. To this, 10 ml of anhydrous toluene in which 0.02mmol of tetraethoxysilane and 0.02 mmol of APTS had been dissolved wasadded, and the mixture was allowed to be reacted for 15 hours. Afterbeing collected by centrifugation, dispersion to ethanol was attempted.After 5 hours of still standing, the supernatant liquid was separated bydecantation. The separated supernatant liquid was centrifuged and thesolid content was dispersed to ethanol/water (1:1). Centrifugation anddispersion to water of pH 3.5 were attempted. As agglutinate was hardlyobserved at this stage, it was confirmed that LaF₃: Yb,Er/(APTS-containing layer) fine particles had been produced. The abovewas collected by centrifugation and vacuum-dried at the room temperaturefor 24 hours. The weight after being dried was 4.1 g. When semiconductorlaser beam of 980 nm was irradiated thereon, it emitted light of redcolor. From the SEM observation, it was confirmed that the averageparticle diameter was approximately 32 nm.

1. A fine particle containing a rare earth element, which is excited bylight having a wavelength in a range of 500 nm to 2000 nm and therebyemits up-conversion emission, wherein the fine particle containing arare earth element comprises: a core portion containing the rare earthelement; and a functional shell portion modifying a surface of the coreportion; wherein the functional shell portion comprising at least aspecific binding substance binding site which can be bound to a specificbinding substance, and wherein the functional shell portion furthercomprises an agglutinate preventing site which prevents agglutination ofthe fine particles containing a rare earth element, and wherein theagglutinate preventing site is at least one functional group selectedfrom the group consisting of ethylene oxide site, phosphate ester site,betaine site, sulphonic group and imino group.
 2. The fine particlecontaining a rare earth element according to claim 1, wherein thespecific binding substance binding site is at least one of, a site withcondensation reactivity, a site with addition reactivity, a site withsubstitution reactivity, or a site capable of effecting a specificinteraction.
 3. The fine particle containing a rare earth elementaccording to claim 1, wherein the agglutinate preventing site is atleast one of a site having hydrogen bonding, or a site with hydratingability.
 4. The fine particle containing a rare earth element accordingto claim 1, wherein the functional shell portion comprises a coreportion binding site, which binds to the core portion.
 5. The fineparticle containing a rare earth element according to claim 4, whereinthe core portion binding site is at least one of, a site with a functionto bind to a metal, a site with condensation reactivity, a site withaddition reactivity, or a site with substitution reactivity.
 6. The fineparticle containing a rare earth element according to claim 1, whereinthe average particle diameter of the core portion is in a range of 1 to100 nm.
 7. The fine particle containing a rare earth element accordingto claim 1, wherein the core portion is made of a halide or an oxide asa parent material, and comprises a rare earth element which can emit theup-conversion emission.
 8. The fine particle containing a rare earthelement according to claim 1, wherein the rare earth element is at leastone of rare earth element selected from the group consisting of erbium(Er), holmium (Ho), praseodymium (Pr), thulium (Tm), neodymium (Nd),gadolinium (Gd), europium (Eu), ytterbium (Yb), samarium (Sm) and cerium(Ce).
 9. The fine particle containing a rare earth element according toclaim 1, wherein the fine particle containing a rare earth element is afine particle of 1 to 100 nm average particle diameter of an oxide dopedwith the rare earth element, and the oxide is at least one kind selectedfrom the group consisting of yttrium oxide, gadolinium oxide, lutetiumoxide, lanthanum oxide and scandium oxide.
 10. The fine particlecontaining a rare earth element according to claim 9, wherein the rareearth element is erbium and ytterbium.
 11. The fine particle containinga rare earth element according to claim 9, wherein the rare earthelement is erbium.
 12. The fine particle containing a rare earth elementaccording to claim 9, wherein the rare earth element is thulium andytterbium.
 13. A fluorescent probe comprising; the fine particlecontaining a rare earth element according to claim 1; and a specificbinding substance which binds to the fine particle containing a rareearth element.
 14. A fluorescence probe according to claim 13, whereinthe specific binding substance is any one of, polynucleotide, hormone,protein, antigen, antibody, peptide, cell or tissue.